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Establishing single-molecule localization microscopy as a quantitative tool towards structural cell biology.

Thevathasan, Jervis Vermal

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In my PhD, I set out to establish single molecule localization microscopy (SMLM) as a complementary technique to answer questions in structural cell biology. The strengths of SMLM are resolution in the nanometer regime, molecular specificity and the ability to record dynamic information.

In this thesis, I report on two independent projects: 1. The use of nuclear pores as a versatile reference standard for quantitative superresolution microscopy. 2. Visualizing the self-assembly of alpha-synuclein fibril polymorphs.

In the first project, we introduced to the field nuclear pore complexes (NPC) as a reference standard for quality control in superresolution microscopy. To this end we generated, four CRISPR cell lines with nucleoporin96 (Nup96) endogenously tagged with labels SNAP, Halo, mEGFP and mMaple. The success of NPCs as a reference standard is owed to its well characterized structural organization and composition. Nup96 is present as 32 copies divided equally over the cytoplasmic and nuclear ring of the NPC. It’s stereotypic arrangement facilitates the visualization of the NPC’s radial eightfold symmetry under SMLM. The overall dimensions of the NPC positions fluorophores at relevant distances for 2D and 3D resolution calibration and quality control. Knowledge of the absolute number of underlying labels present in each NPC allowed us to calculate the effective labeling efficiency of each labeling strategy. Having a defined number for fluorophores also allowed for counting of protein copy numbers within complexes using both diffraction-limited and superresolution microscopy.

In the second project, I established a correlative transmission electron microscopy (TEM) and single molecule localization microscopy (SMLM) method to study the dynamic self-assembly of amyloid fibril polymorphs. Amyloid fibril polymorphism has been found in distinct neurodegenerative disease phenotypes. The ability to exist as different polymorphs has been a stumbling block towards understanding disease etiology. To address this need, I first established an imaging assay that enabled the visualization of the self-assembly process of single amyloid fibrils in real-time. To visualize individual fibrils, I used the point accumulation for imaging in nanoscale topography (PAINT) imaging strategy with fluorogenic amyloid binding dyes. This strategy allowed for imaging with unmodified protein monomers while achieving high labeling densities on fibrils permitting the characterization of respective fibril self-assembly. From my dynamic PAINT measurements, I have identified that fibrils exhibit growth characteristics specific to the solution conditions they are in. The unique opportunity of analyzing the polarization of the emitted fluorescence of each binding event enabled me to visualize fibril ultrastructure. To further validate observed structural features, I performed correlative TEM tomography and dynamic PAINT. Such multiparametric correlative imaging enables the description of fibril growth kinetics with respect to its underlying structure, which would otherwise not be possible.

Item Type: Dissertation
Supervisor: Ries, Dr. Jonas
Place of Publication: Heidelberg
Date of thesis defense: 15 October 2019
Date Deposited: 11 Nov 2019 09:09
Date: 2020
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
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